Mapping CAC susceptibility loci to atherosclerotic plaques

Project ID: AE_20200512_COL_MKAVOUSI_MBOS_CHARGE_1000G_CAC.

Background

Collaboration for mapping targets from the CHARGE Consortium 1000G GWAS on coronary artery calcification (CAC) with Maryam Kavousi, Patricia Peyser, and Maxime Bos.

Athero-Express Biobank Study

We have bulk RNAseq (n = 635 samples) and single-cell RNAseq data, genome-wide methylation (Illumina 450K) in n ± 600, as well as overlapping genetic data for ±2,100 individuals with extensive histological plaque characterisation. We will address the following questions:

  • Gene expression correlated to characteristics of plaques?
  • Where target genes expressed, which cell types?
  • Are any of the variants associated to plaque characteristics?

Ideally, we would like to map the variants to the expression and methylation data (molecular QTL mapping), but this is pending finalizing our molQTL mapping.

We expect new plasma-based OLINK data (for the ‘TO_AITION’ projecty) in Q4 2020. We agreed that we would keep this in mind, we could run some additional analyses when asked during the revision stage of the manuscript.

  • Correlation of variants to proteins from the OLINK-platform

Methods

Mapping genes to single cells

We will use the last dataset from the scRNAseq data, including 35 individuals, to project target genes.

Genetic analyses

For the genetic analyses we will perform regression analyses adjusted for age, sex (where applicable) and principal components. So, we will apply the following model:

model 1: phenotype ~ age + sex + chip-used + PC1 + PC2 + year-of-surgery

phenotypes are:

  • calcification, coded Calc.bin no/minor vs. moderate/heavy staining
  • collagen, coded Collagen.bin no/minor vs. moderate/heavy staining
  • fat10, coded Fat.bin_10 no/<10% fat vs. >10% fat
  • fat40, coded Fat.bin_40 no/<40% fat vs. >40% fat
  • intraplaque hemorrhage, coded IPH.bin no vs. yes
  • macrophages (CD68), coded macmean0 mean of computer-assisted calculation CD68+ region of interest
  • smooth muscle cells (alpha-actin), coded smcmean0 mean of computer-assisted calculation SMA+ region of interest
  • intraplaque vessel density (CD34), coded vessel_density manually counted CD34+ cells per 3-4 hotspots
  • mast cells, coded Mast_cells_plaque manually counted mast cell tryptase+ cells (https://academic.oup.com/eurheartj/article/34/48/3699/484981)
  • neutrophils (CD66b), coded neutrophils manually counted CD66b+ cells (https://pubmed.ncbi.nlm.nih.gov/20595650/)

Continuous variables were inverse-rank normal transformated, indicated by _rankNorm.

Top hits are loci on chromosome 9, 10 and 19 associated to mast cells, calcification, and fat40.

Phenotype VariantID Chromosome Basepair Other allele Coded allele Total N MAC MAF CAF HWE p-value P-value Effect size (beta) standard error (SE)
Calcification 10:44511785 10 44511785 C A 1501 617 0.206 0.794 0.082 0.040 0.201 0.098
Calcification 10:75917431 10 75917431 A G 1501 848 0.282 0.718 0.308 0.005 0.237 0.085
Calcification 6:12919867 6 12919867 G A 1501 1157 0.385 0.615 0.957 0.034 0.167 0.079
Calcification 6:12921714 6 12921714 A T 1501 1049 0.349 0.349 0.650 0.013 -0.201 0.081
Collagen 19:45382675 19 45382675 G A 1497 97 0.032 0.032 1.000 0.034 -0.501 0.230
Collagen 19:45412079 19 45412079 C T 1497 182 0.061 0.061 0.064 0.038 -0.359 0.169
Collagen 7:45960645 7 45960645 G C 1497 1175 0.392 0.392 0.304 0.043 0.187 0.093
Fat40 19:45382675 19 45382675 G A 1503 96 0.032 0.032 1.000 0.046 0.466 0.229
Fat40 19:45412079 19 45412079 C T 1503 181 0.060 0.060 0.062 0.006 0.450 0.161
IPH 6:12919867 6 12919867 G A 1500 1161 0.387 0.613 0.957 0.035 -0.170 0.081
SMC_rankNorm 9:21995882 9 21995882 C T 1396 1363 0.488 0.488 0.181 0.036 -0.074 0.035
SMC_rankNorm 9:22067593 9 22067593 A G 1396 1194 0.428 0.428 0.022 0.038 0.075 0.036
SMC_rankNorm 9:22072638 9 22072638 G A 1396 1363 0.488 0.488 0.006 0.013 0.089 0.036
SMC_rankNorm 9:22085598 9 22085598 T C 1396 1340 0.480 0.480 0.005 0.019 0.084 0.036
SMC_rankNorm 9:22124744 9 22124744 C G 1396 1389 0.497 0.497 0.087 0.027 0.078 0.035
MAC_rankNorm 10:44511785 10 44511785 C A 1401 577 0.206 0.794 0.102 0.023 -0.101 0.044
Neutrophils_rankNorm 15:79123946 15 79123946 G T 238 187 0.393 0.607 0.892 0.041 0.183 0.089
Neutrophils_rankNorm 6:12921714 6 12921714 A T 238 171 0.359 0.359 0.573 0.040 -0.182 0.088
MastCells_rankNorm 9:22056359 9 22056359 A G 205 168 0.410 0.590 0.773 0.006 -0.287 0.104
MastCells_rankNorm 9:22067593 9 22067593 A G 205 176 0.429 0.429 0.201 0.034 -0.223 0.104
MastCells_rankNorm 9:22068652 9 22068652 G A 205 154 0.376 0.624 0.655 0.002 -0.325 0.106
MastCells_rankNorm 9:22071751 9 22071751 G A 205 161 0.393 0.607 0.379 0.004 -0.312 0.106
MastCells_rankNorm 9:22072638 9 22072638 G A 205 203 0.495 0.495 0.404 0.042 -0.210 0.103
VesselDensity_rankNorm 19:45412079 19 45412079 C T 1302 155 0.060 0.060 0.128 0.023 0.174 0.077
VesselDensity_rankNorm 6:12768218 6 12768218 C T 1302 417 0.160 0.160 0.537 0.026 -0.114 0.051
VesselDensity_rankNorm 6:12801967 6 12801967 A C 1302 1034 0.397 0.397 0.729 0.030 -0.083 0.038
VesselDensity_rankNorm 6:12919867 6 12919867 G A 1302 1006 0.386 0.614 0.907 0.050 -0.075 0.038
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Gene expression

Whole-plaque RNAseq

For the expression analysis we used carotid plaque-derived bulk RNAseq data and queried it for the gene list. Below a graph showing the overall expression of the genes (not all are in the data) compared to the mean expression of 1,000 randomly picked genes.

Figure 1: Overall expression of target genes in carotid plaques from the Athero-Express Biobank Study Overall expression of target genes in carotid plaques from the Athero-Express Biobank Study

We assessed the correlation with plaque characteristics (mentioned above) and secondary major adverse cardiovascular events (MACE [major]) at 30 days and 3 years after CEA.

Single cell RNAseq

We projected target genes to the single-cell RNAseq data derived from 37 carotid plaque samples. We identified cell communities (Figure 2), mapped and projected target gene expression to the cell communities (Figure 3).

Figure 2: Cell communities identified in carotid plaques from the Athero-Express Biobank Study Cell communities identified in carotid plaques from the Athero-Express Biobank Study

Figure 3: Dotplot showing expression of target genes per cell type in carotid plaques from the Athero-Express Biobank Study Dotplot showing expression of target genes per cell type in carotid plaques from the Athero-Express Biobank Study

QTL mapping

Quantitative trait loci mapping has not finished. This could be added at a later stage.

The MIT License (MIT)